Plant Physiol. (1988) 87, 320-3240032-0889/88/87/0320/05/$01 .00/0

31P and 13C-NMR Studies of the Phosphorus and CarbonMetabolites in the Halotolerant Alga, Dunaliella salina1

Received for publication September 28, 1987 and in revised form January 19, 1988

MICHAL BENTAL, MICHAL OREN-SHAMIR, MORDHAY AVRON*, AND HADASSA DEGANIDepartments of Isotope Research (M.B., H.D.) and Biochemistry (M.S., M.A.), The Weizmann Instituteof Science, Rehovot 76100, Israel

ABSTRACT

The intracellular phosphorus and carbon metabolites in the halotolerantalga Dunaliella salina adapted to different salinities were monitored inliving cells by 31P- and "3C-nuclear magnetic resonance (NMR) spectros-copy. The 13C-NMR studies showed that the composition of the visibleintracellular carbon metabolites other than glycerol is not signiflcantlyaffected by the salinity of the growth medium. The T1 relaxation rates ofthe 13C-glycerol signals in intact cells were enhanced with increasing sa-linity of the growth medium, in parallel to the expected increase in theintracellular viscosity due to the increase in intracellular glycerol. The31P-NMR studies showed that cells adapted to the various salinities con-tained inorganic phosphate, phosphomonoesters, high energy phosphatecompounds, and long chain polyphosphates. In addition, cells grown inmedia containing up to 1 molar NaCI contained tripolyphosphates. Thetripolyphosphate content was also controlled by the availability of inor-ganic phosphate during cell growth. Phosphate-depleted D. salina con-tained no detectable tripolyphosphate signal. Excess phosphate, however,did not result in the appearance of tripolyphosphate in 31P-NMR spectraof cells adapted to high (>1.5 molar NaCI) salinities.

Members of the genus Dunaliella (division Chlorophycophyta,order Volvocales, family Polyblepharidaceae) are unicellular,motile, green algae, which lack a rigid cell wall. Dunaliella hasthe capacity to tolerate and adapt to a wide range of salt con-centrations (0.1-5.5 M NaCl). This is achieved through the abilityof the algae to survive the initial osmotic stress, and then adjusttheir intracellular osmotic content to the new required level, bysynthesis or elimination of glycerol (3, 4).

Little is known about the intracellular phosphate compoundsin Dunaliella, and no characterization of the intracellular phos-phate compounds is available. Ginzburg and Ginzburg (9) meas-ured an intracellular inorganic phosphate concentration of 400mm for 12 species of Dunaliella, cultured at 0.5 or 2 M NaCl and2 mm phosphate. Pick et al. (20) showed that the cells accumulatephosphate in proportion to its concentration in the growth me-dium. Gimmler and Moller (8) studied the phosphates of D.parva cells following osmotic shocks. Following a hyperosmoticshock, they observed a very rapid increase in the endogenousinorganic phosphate level, and in the level of an unidentifiedphosphate compound, at the expense of the acid-soluble, cellularorganic phosphates. A hypoosmotic shock caused a rapid de-crease in the intracellular inorganic phosphate level. The ATPto ADP ratio, the phosphorylation potential (ATP/ADP x Pi),

I Supported by a grant from the United States/Israel Binational ScienceFoundation.

and the 3-phosphoglyceric acid to Pi ratio also decreased.In general, algal intracellular phosphate levels can vary widely,

depending on whether the algae are growing under phosphorus-rich or phosphorus-limited conditions (11). Like other micro-organisms, algal cells possess the ability to accumulate excessphosphate. This is stored in the form of condensed phosphates,which can be either cyclic (metaphosphates) or linear unbranchedchains (polyphosphates). The existence of intracellular poly-phosphate granules, whose size (30-500 nm) and number wereaffected by the growth conditions, was demonstrated in the algaeChlorella pyrenoidosa (19) and Anabaenaflos-aquae (21) by elec-tron microscopic studies combined with x-ray energy dispersiveanalysis.

In this paper, we report the use of 31P-NMR spectroscopy tocharacterize the intracellular phosphate metabolites in living D.salina cells adapted to different salinities and phosphate concen-trations. In vivo 31P-NMR has been used extensively in studiesof mammalian cells and organs, including organs in living animals(2, 7 and references therein), and recently also in studies ofunicellular photosynthetic organisms (10, 14, 15, 18, 24, 25).We also report the use of '3C-NMR spectroscopy to study D.

salina grown in media containing '3C-enriched carbonate andmonitored under conditions similar to those used in the 31p stud-ies. 13C-NMR was employed previously in numerous studies ofintact cells and organs (1). The glycerol in D. salina grown in2.1 M NaCl was previously monitored by natural abundance 13C-NMR studies (17). '3C-labeled D. salina permitted additionalcharacterization of several other intracellular metabolites (5). Inthe latter study, it was also shown that following a hypoosmoticshock, glycerol is converted to a(1-*4)glucan, and the reverseprocess occurs following a hyperosmotic shock. In this study wemonitored, by in vivo '3C-NMR, the intracellular glycerol, me-tabolites, and membranal components in cells adapted to a widerange of salinities.

MATERIALS AND METHODS

Growth Conditions. Dunaliella salina was grown in batch cul-tures in media containing NaCl at the indicated concentration,50 mM NaHCO3, 5 mm KNO3, 5 mM MgSO4, 0.3 mm CaCl2, 0.8/1M ZnC12, 0.02 /.M CoCl2, and 0.2 nM CuCl2 (initial pH, 7.5-8.2). Algal cultures were grown under continuous illuminationwith white fluorescent lamps (light intensity 3800 lux) at 26°Cwith slow (80 rpm) continuous shaking within a New BrunswickPsycrotherm incubator. Adaptations to different NaCl or phos-phate concentrations were made by growing for several daysunder the desired conditions. Cells for 13C-NMR studies werecultured in the same medium as above, except that it contained25 mM [13C, 10%]NaHCO3 for the last 2 to 3 division cycles.

Preparation of Cells for NMR Measurements. Algae at thelogarithmic phase (1-2.5 x 106 cells/mL) were concentrated bycentrifugation at 4°C (2000g for 10 min). All subsequent oper-

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PHOSPHATE AND CARBON METABOLITES IN DUNALIELLA

ations were performed at 0 to 4°C. Cell number was determinedin a Coulter Counter. For the NMR experiments, cells werewashed once and resuspended in medium containing NaCl andKH2PO4 at the indicated concentrations, 50 mm NaHCO3, 5 mMKNO3, 5 mm MgSO4, and 0.3 mm CaCl2 (pH 8.2). In all exper-iments, the final cell suspension (1-2 mL of 2 x 109 cells/mL)was transferred to a 10 mm NMR tube and was immediatelyaerated.NMR Measurements. 31P- and '3C-NMR experiments were

conducted at 4 + 1°C and 12 + 1°C, respectively (unless indi-cated otherwise). The cells were aerated during the NMR meas-urements by passing humidified air through silicon tubing im-mersed in the cell suspension: the tubing allows gas exchangebut is impermeable to the medium.NMR measurements were performed with a Bruker CXP-300

FT NMR spectrometer. 31P-NMR spectra were recorded at 121.5MHz, using 600 pulses and a repetition time of 1 s, or 900 pulsesand a repetition time of 7 s. An external reference containing 1M H3PO4, 0.88 M HCl, and 113 mm PrCl3 as a shift reagent wasused in some of the experiments. Assignments of the 31P reso-nances were established by addition of the commercially avail-able compounds to an extract of the cells. '3C-NMR spectra wererecorded at 75.47 MHz, using 450 pulses and a repetition timeof 1 s, with broadband proton decoupling (= 1 W). The intra-cellular glycerol served as an internal chemical shift reference.The assignment of the 13C resonances was based on previousstudies (5).

T1 measurements were performed by using the inversion re-covery method, applying 180'-r-900 pulses. Each measurementof T, of the algal phosphate signals included 9 variable r values,from 0.01 to 6 s, and a 6 s repetition time. Each measurementof T, of the algal glycerol '3C signals included 19 variable T values,from 0.002 to 10 s, and a 10 s repetition time. T, values weredetermined from a nonlinear exponential fit (23) of the mag-netization recovery versus the delay (T) curve.NOE2 of the glycerol resonances was determined by comparing

intensities in spectra recorded with complete 'H decoupling (900pulse and 10 s repetition time) with those in spectra recordedwith 'H gated decoupling, i.e. decoupling applied during thepulse only. To maintain the same temperature (within 2°C)throughout the experiment, 8 scans were accumulated with fulldecoupling and then 8 scans were accumulated with gated de-coupling; this sequence was repeated 8 times.

RESULTS AND DISCUSSION

31P-NMR Studies of the Phosphate Metabolites. The stabilityof cellular metabolism under the conditions of the NMR exper-iment was established by observation of the cells' motility andshape under the microscope and by following the phosphatemetabolites. The composition and content of the phosphoruscompounds reflect the energy state of the cell, the high energymetabolites being a most sensitive monitor of the cell's viability.

Figure 1 is a fully relaxed 31P-NMR spectrum of D. salinagrown with 1 M NaCl and 0.2 mm phosphate (standard growthconditions). Under these conditions, the following prominentresonances appear in the 31P NMR spectrum: (a) phosphomon-oesters (PME), including glycerol phosphate and sugar phos-phates; (b) inorganic phosphate in the growth medium (Piext);(c) intracellular inorganic phosphate (Pii0t); (d) an unidentifiedpeak (X); (e) two large signals of tripolyphosphate-one due tothe two terminal phosphate groups (PPP) and the other, at ahigher field, due to the middle phosphate (PPP); (f) NTP signalsdue to a- and 13-NTP. The y-NTP and ,8-NDP overlap the largePPP signal. The resonance denoted a-NTP may also include

2 Abbreviations: NOE, nuclear Overhauser enhancement; NTP, nu-cleoside triphosphate; NDP, nucleotide diphosphate.

PPiintXP iext\ I

PpPY-NTP/3-NDPE4P*nP

II

10 5 0 -5 -10 -15 -20 -25ppm

FIG. 1. Fully relaxed 3'P-NMR spectrum of D. salina cells grown with1.0 M NaCI, 0.2 mm phosphate. Cells were prepared as described under"Materials and Methods" and maintained at 4°C. The spectrum wasobtained by accumulating 592 scans (900 pulses, 7 s delay) and processedusing line broadening of 10 Hz. For peak assignments, see text. Ext.ref., external reference. Chemical shift of 0 ppm refers to 85% H3PO4.

signals due to nicotinamide adenine dinucleotides and a-NDP;(g) a signal due to the middle phosphate residues of medium size(4-10 residues) polyphosphate chains (P-(P)-P). The signal ofthe terminal phosphate residues of these polyphosphates alsooverlaps that of the terminal phosphates of tripolyphosphate.31P-NMR studies of the freshwater unicellular green algae

Chlorella (14, 18, 24), Cosmarium (6), and Scenedesmus (22)and of the marine flagellate Heterosigma akashiwo (26) haverevealed the presence of intracellular NTP, sugar phosphates,and polyphosphate chains. Unlike in Dunaliella, however, nosignals due to tripolyphosphate were observed in intact cells ofany of them. In Scenedesmus (22) and Chlorella (24), an uni-dentified resonance was observed, with a chemical shift similarto the resonance designated X (Figs. 1 and 2). Further studiesare necessary for identification of this signal, as well as for de-tailed assignment of the sugar phosphate resonances.

T, values of the identified intracellular phosphate metabolitesranged between 50 ms (for tripolyphosphate) and 500 ms (forPi). The unassigned signal X had a T, of approximately 1 s. Thus,except for X, there were no saturation effects in the 31p spectrarecorded with 60° pulses and 1 s repetition time. Under theseconditions, a spectrum with a good signal to noise ratio couldbe obtained within 30 min (Figs. 2-4).The concentration of the phosphates, determined from 31P

NMR spectra of cells maintained at 4°C, remained stable for atleast 6 h. At 20°C, minor changes were observed during the first2 to 3 h of measurements. Upon prolonged (overnight) incu-bation at 35°C without air flow, large changes were observed;the spectrum became dominated by very intense, broad peaksof Pi and medium size polyphosphate chains (see Figs. 4, B andD, and 3C), and the total integrated intensity increased several-fold. These changes reflect the presence of high concentrationsof NMR-invisible phosphates in the living cells, probably storedas insoluble, long-chain polyphosphate deposits that are hy-drolized into shorter chains and free inorganic phosphate.

Effects of Salinity and Phosphate Availability on the Intracel-lular Phosphate Metabolites. Examination of cells adapted todifferent NaCl concentrations revealed that cells grown at 1 MNaCl or lower (0.25 M, 0.5 M) exhibited a spectrum similar tothat shown in Figure 2A. At higher salinities (1.5, 2, 4 M NaCl),however, the amount of tripolyphosphate decreased to a non-detectable level (Figs. 2B and 3A). The tripolyphosphates werenot found in cells cultured at high salinities even when excess

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A

BP,nt

Pient

PPPx Y- NTP

-f l 3-NDPP lex I - P tn P

P4P}n P

P'i Int

APlext

PME

ext. ref.

NTPP4P

L~~~~~~VA~~~~~4~

C

ext rY X

P{P),, P

Pf*PnP

Y-NDP

II I I I10 5 0 -5 -10 -15 -20 -25 -30

ppm

FIG. 2. 3IP-NMR spectra of D. salina adapted to different salinities.Cells were prepared as described under "Materials and Methods" andmaintained at 4°C. Each trace was obtained by accumulating 1800 scans

(30 min) (60° pulses, 1 s delay) and processed using line broadening of15 Hz. 0 ppm refers to 85% H3P04. A, Cells grown with 1.0 M NaCl;B, cells grown with 2.0 M NaCl.

phosphate was available (Fig. 3B). However, these cells did havelong-chain polyphosphates, as is apparent from the polyphos-phate signal that appeared upon overnight incubation withoutaeration at 35°C (Fig. 3C).

Cells grown with excess phosphate (1 mM) throughout thesalinity range showed the same spectral features as cells culturedat 0.2 mm phosphate (e.g. Fig. 3, A and B for cells cultured at4 M NaCl). When cells were cultured under phosphate-limitedconditions (0.03 mM), the tripolyphosphate signals disappeared,and the intracellular Pi-to-ATP ratio increased (Fig. 4A versus

Fig. 4C). The resulting 31P NMR spectrum was reminiscent ofspectra obtained for cells cultured at high salt. However, in con-

trast to the cells adapted to high salinities, upon overnight unaer-

ated incubation of the phosphate-depleted cells at 35°C, only a

relatively small signal of inorganic phosphate appeared, and no

signals due to polyphosphates (Fig. 4B versus Fig. 4D). Thissuggests that the content of stored polyphosphates in these cellswas very low.These results suggest that the presence of tripolyphosphate in

the cells is controlled by both the phosphate availability (as are

the long-chain polyphosphates) and the salinity of the growth

IOppm

FIG. 3. -'P-NMR spectra of D. salina grown with 4.0 M NaCI: effectof phosphate availability. Experimental conditions as described for Fig-ure 2. A, Cells cultured with 0.2 mm phosphate, maintained at 4°C; B.cells cultured with 1.0 mm phosphate, maintained at 4°C; C, the cells of(B), following overnight unaerated incubation at 35°C. Y, an unassignedsignal.

medium. The significance of this finding is not yet clear.The accumulation of excess phosphate and its storage in the

form of high mol wt, condensed, cyclic metaphosphates or linearpolyphosphates by algal cells is a well known phenomenon. Itwas shown (11) that in algae (and in other microorganisms) syn-thesis of polyphosphates occurs when the energy requiring re-actions are saturated or in relative excess due, for example, tolack of CO2. The general conclusion from numerous studies onthe interrelations between phosphorus metabolism and photo-synthesis is that in algae polyphosphates are synthesized in thelight at the expense of ATP generated by photophosphorylationreactions. They are thought to function not as an energy reservebut as a phosphorus reserve which enables the cells to grow underphosphorus depletion. Such a role was also proposed for poly-phosphates of bacteria and yeast (13). It has been suggested (16)that in algae short (2-7 residues) oligopolyphosphates are firstsynthesized and then are condensed to high mol wt phosphates(200-300 residues) and mostly converted to metaphosphates. Ofthe latter, trimetaphosphates regulate inorganic orthophosphateuptake via inositol phosphates; therefore, the ATP-ADP-Pi sys-tem, the condensed phosphates, and the inositol phosphates areclosely related. In the 31P-NMR spectrum, the signals due to thephosphate groups of the inositol phosphates would be in the areaof the inorganic phosphates and sugar phosphates (2-4 ppm)which we cannot yet resolve.'3C-NMR Studies of the Carbon Metabolites. '3C-NMR spectra

were recorded for cells grown in media containing 0.1, 0.5, 1,2, 3, and 4 M NaCl. Typical spectra are shown in Figure 5. Aspreviously noted (3, 5), the difference in salinity markedly af-fected the concentration of glycerol. It may be noted that glycerolcontent in cells adapted to a low salt concentration (0.1 M NaCI)

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PHOSPHATE AND CARBON METABOLITES IN DUNALIELLA

B Pi

Pi

D

P4PnP

p4pEnp

10 ppm

FIG. 4. 3'P-NMR spectra of D. salina cultured with 1.0 M NaCl: effectof phosphate availability. Experimental conditions as described for Fig-ure 2. A, Cells cultured with 0.03 mm phosphate, maintained at 4°C; B,the cells of (A) following overnight unaerated incubation at 35°C; C,cells cultured with 0.2 mm phosphate, maintained at 4°C; D, the cells of(C), following overnight unaerated incubation at 35°C. Y, Y,-unas-signed signals. The spectrum in (D) is reduced to 1/4 relative to thespectra in (A)-(C).

was very low, in the range of concentrations of the other commonmetabolites (Fig. 5A). However, there are no distinct changesin the composition and concentration of the other NMR-visiblemetabolites. This suggests that although during the adaptationto a new salt concentration the cellular metabolism undergoeslarge changes (5), once the cells are adapted the metabolic in-termediates reach a similar steady state level at all salinities.Some differences appeared in the membranal signals, primarilyin the intensity of (CH2)n that increased at the very high salinities(3 and 4 M NaCl). Further studies are required to characterizethese changes.

Effect of Salinity on T1 and Nuclear Overhauser Enhancementof Glycerol. The T, relaxation times of the '3C-glycerol signalswere measured in intact cells adapted to different salinities aswell as in model solutions containing different glycerol concen-trations (Fig. 6). For glycerol, which is a small molecule thattumbles rapidly, 13C relaxation occurs mainly via dipole-dipoleinteractions between the 13C and the directly bonded protons.The dipolar relaxation rate (1TI) is directly proportional to thecorrelation time for the reorientation of the glycerol molecule(re), according to

1/NT, = 2.0325 x 1011 x rc

where N is the number of directly bonded protons. is pro-portional to the viscosity of the glycerol environment (12). Theincrease in 1/NT, with salinity paralleled the increase observedfor glycerol solutions, except at the very low salinity, and wasin accord with the expected increase in viscosity due to the in-creased glycerol concentration (Fig. 6). The higher values forthe relaxation rates in the cells relative to the glycerol solutions

l I

170 130I I

1090 50ppm

FIG. 5. '3C-NMR spectra of D. salina adapted to different salinities.Cells were prepared as described under "Materials and Methods." Cellsgrown with: A, 0.1 M NaCl; B, 1.0 M NaCl; C, 4.0 M NaCl. Cell densityin (C) was approximately 75% that in (A) and (B). Each trace was

obtained by accumulating 1800 scans (450 pulses, 1 s delay) and processedusing line broadening of 5 Hz. Spectra were recorded at 12°C. LAC,lactate; GLU, glutamate; ALA, alanine; -CH3, -CH2-, -CH =CH-,lipid methyl, methylene, and methene carbons, respectively; -COO-,carboxylate resonances. The chemical shift scale was referenced to te-tramethylsilane (TMS) using the glycerol signals as an internal reference(63.6, 73.1 ppm).

may reflect the higher viscosity in the cells due to the presenceof additional soluble metabolites and macromolecules. This isexpected to be more pronounced at the low salinity, where thecontribution of glycerol to the total viscosity is smaller, explainingthe deviation of the 1/NT, curve at this range.Although the '3C-'H dipolar interaction dominates glycerol

relaxation, other relaxation mechanisms may also prevail, par-ticularly in the cells. A measure for the contribution of the othermechanisms was obtained from the T,s and the NOEs using thesame analysis as Norton et al. (17). These calculations have in-dicated that the contribution of mechanisms other than the di-polar interaction to the T, relaxation is small (10-20%). Thisresult differs from that of Norton et al. (17), who determinedapproximately 40% contribution from mechanisms other thandipolar to the T1 relaxation rate. It therefore makes unlikely a

A

C

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lI

2 3 4

'Tmosity (M)

FIG. 6. Effect of the growth medium osmosity on the T, relaxationrates of the intracellular glycerol in D. salina. The osmosity of a solutionis defined as the molar concentration of an NaCl solution having thesame osmotic pressure. The solid line in (C) represents the change inthe relative viscosity ij/ij, (the viscosity of a solution relative to theviscosity of pure water) of glycerol-water mixtures as a function of theirosmosity (data from [27]). (A---A), the relaxation rate, I/T,, of '3C nucleiof intracellular glycerol in D. salina cells as a function of the mediumsalinities in which they were grown; (- --), represents the relaxationrate, I/T1, of the 13C nuclei of glycerol in glycerol-water mixtures con-

taining increasing glycerol concentration, as a function of their osmosity.

A, '3CH2OH; B, 13CHOH. Each point represents an independent ex-

periment, the error bars delineate the 95% confidence interval obtainedfrom the best-fit procedure as described under "Materials and Methods."

marked paramagnetic effect (17) on the T1 relaxation rate ofintracellular glycerol. We have also found that the T, relaxationdue to the dipolar interaction and due to the other mechanismswas similarly enhanced by increasing salinity of the growth me-

dium (for example, for 13CHOH, 1/NT,(dipolar) increases from 0.7to 1.1 s- 1, and 1/NTl(other) increases from 0.1 to 0.3 in cells grownin 0.5 and 4.0 M NaCl, respectively). This suggests that the mod-ulation of the T, with salinity is caused primarily by the increasedglycerol content of the cell, in line with the results presented inFigure 6.

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